Radioconjugation method

ABSTRACT

The present invention relates to the field of radiopharmaceuticals for in vivo imaging, in particular to a method of labelling a biological targeting molecule with a radioisotope. The method of the invention is particularly suitable for use with an automated synthesizer apparatus. Also provided are precursors in sterile form, as well as cassettes comprising such precursors useful in the method.

FIELD OF THE INVENTION

The present invention relates to the field of radiopharmaceuticals forin vivo imaging, in particular to a method of labelling a biologicaltargeting molecule with a radioisotope. The method of the invention isparticularly suitable for use with an automated synthesizer apparatus.Also provided are precursors in sterile form, as well as cassettescomprising such precursors useful in the method.

BACKGROUND TO THE INVENTION

WO 2004/080492 A1 discloses a method for radiofluorination of abiological targeting vector, comprising reaction of a compound offormula (I) with a compound of formula (II):

or,a compound of formula (III) with a compound of formula (IV)

wherein:

-   -   R1 is an aldehyde moiety, a ketone moiety, a protected aldehyde        such as an acetal, a protected ketone, such as a ketal, or a        functionality, such as diol or N-terminal serine residue, which        can be rapidly and efficiently oxidised to an aldehyde or ketone        using an oxidising agent;    -   R2 is a group selected from primary amine, secondary amine,        hydroxylamine, hydrazine, hydrazide, aminoxy, phenylhydrazine,        semicarbazide, and thiosemicarbazide and is preferably a        hydrazine, hydrazide or aminoxy group;    -   R3 is a group selected from primary amine, secondary amine,        hydroxylamine, hydrazine, hydrazide, aminoxy, phenylhydrazine,        semicarbazide, or thiosemicarbazide, and is preferably a        hydrazine, hydrazide or aminoxy group;    -   R4 is an aldehyde moiety, a ketone moiety, a protected aldehyde        such as an acetal, a protected ketone, such as a ketal, or a        functionality, such as diol or N-terminal serine residue, which        can be rapidly and efficiently oxidised to an aldehyde or ketone        using an oxidising agent;        to give a conjugate of formula (V) or (VI) respectively:

wherein X is CO—NH—, —NH—, —O—, —NHCONH—, or —NHCSNH—, and is preferably—CO—NH—, —NH— or —O—; Y is H, alkyl or aryl substituents; andthe Linker group in the compounds of formulae (II), (IV), (V) and (VI)is selected from

wherein:n is an integer of 0 to 20;m is an integer of 1 to 10;p is an integer of 0 or 1;

Z is 0 or S.

Poethko et al [J.Nucl.Med., 45(5), 892-902 (2004)] disclose a method ofradiolabelling peptides with the radioisotope ¹⁸F, wherein anaminooxy-functionalised peptide is condensed with[¹⁸F]-fluorobenzaldehyde to give a labelled peptide having an oximeether linkage as follows:

Schottelius et al [Bioconj.Chem., 19(6), 1256-1268 (2008)] furtherdeveloped the method of Poethko et al. Schottelius et al use anaminooxy-functionalised peptide wherein the amine of the aminooxy groupis protected with an N-Boc (Boc=tert-butyloxycarbonyl) protecting group.The desired aminooxy-functionalised peptide is generated in situ in thepresence of [¹⁸F]-fluorobenzaldehyde via deprotection of the N-Boc groupat acidic pH (pH=2) at 75° C. Schottelius et al use a 5-fold molarexcess of the Boc-protected precursor, because the deprotection was notquantitative under the reaction conditions.

Mezo et al [J.Pept.Sci., 17, 39-46 (2010)] describe some of the problemsassociated with the above oxime ligation chemistry of Boc-protectedaminooxy-functionalised peptides. Thus, it is known that the initiallyformed free aminooxy-peptide can acylate unreacted Boc-protectedaminooxy-peptide, leading to undesirable by-products. It is also knownthat the reactivity of the free aminooxy group of the functionalisedpeptide is high towards carbonyl compounds. Consequently, unwantedcondensation can occur with any adventitious aldehydes or ketonespresent either in the reaction mixture or in any subsequent purificationsteps. Such aldehydes or ketones could be traces of acetone present inthe solvents used, or formaldehyde (eg. from plasticizers). Mezo et alare interested in solving this problem for both the conjugation ofanti-cancer drugs and of [¹⁸F]-fluorobenzaldehyde to peptides. Mezo etal solve the problem by carrying out the deprotection of theBoc-aminooxy peptide in the presence of a tenfold molar excess of free(aminooxy)acetic acid (Aoa) as a ‘carbonyl capture agent’. Thedeprotected aminooxy-peptide and excess Aoa is then lyophilised andstored at 4° C. Immediately prior to the oxime ligation reaction, thelyophilised mixture is reconstituted, and excess Aoa is separated byHPLC or Sep-Pak plus C18 cartridge. Mezo et al provide an example inwhich non-radioactive (i.e. ¹⁹F) 4-fluorobenzaldehyde is conjugated toan aminooxy-functionalised somatostatin peptide using this technique.Mezo et al do not provide any data on ¹⁸F-radiolabelling.

There is therefore still a need for improved methods of radiolabellingpeptides and other biological targeting molecules.

THE PRESENT INVENTION

The present invention provides an improved method for radiolabelling abiological targeting molecule (BTM) via aminooxy functional groups. Theinvention provides a protecting group approach which overcomes theimpurity problems recognised in the prior art without the need for:

-   -   (i) a ‘carbonyl capture agent’ together with prolonged storage        at 4° C.;    -   (ii) strongly acidic conditions to remove the N-Boc aminooxy        protected protecting group;    -   (iii) use of an excess of N-Boc aminooxy protected starting        material.

Option (i) is not attractive for radiolabelling purposes, since theexcess carbonyl capture reagent must be fully removed prior to preventside-reactions involving the carbonyl capture reagent. That removal steprequires preparative chromatography or similar. In addition, thenecessity for storage of the precursor at 4° C. is less than ideal.

The prior art also teaches that a Boc-protected aminooxy precursor canbe used option (ii). The problem with that approach is that stronglyacidic conditions are required for the Boc deprotection, such as 95%aqueous trifluoroacetic acid (TFA) or 25-50% TFA/organic solvent (e.g.DCM), both at room temperature. Some publications also suggest heatingat 60-75° C. Such strong acid and/or heating may damage the biologicaltargeting molecule. It is also necessary to remove the excess strongacid in an additional step typically by evaporation of the TFA, whichcan be time-consuming. The trifluoroacetic acid can, however, alsogenerate salts which would not be removed via evaporation and typicallyrequire trituration to remove.

The method of the present invention achieves complete deprotection usingonly aqueous dilute acid (e.g. 2.5% aqueous TFA or 0.01 M aqueoushydrochloric acid) at room temperature, or 0.1% aqueous TFA at 60° C.Such conditions are more compatible with BTMs, and obviate the need foradditional purification steps.

Thirdly, the prior art teaches that a 5-fold molar excess of aBoc-protected aminooxy-BTM precursor should be used option (iii). Thatis because the Boc-deprotection is incomplete and is it important toconsume all of the [¹⁸F]-fluorobenzaldehyde reactant. That approach isundesirable for radiopharmaceutical purposes, because any excess ofunlabelled BTM precursor present in the product would probably competewith the radiolabelled BTM for the site of biological interest in vivo,and hence risk inhibiting uptake of the desired imaging agent.Consequently, it would be necessary to remove the excess non-radioactiveprecursor from the radiolabelled product prior to use. In contrast, themethod of the present invention permits efficient, complete deprotectionunder mild conditions, so that it is unnecessary to employ an excess ofthe protected starting material.

The protected BTMs of the present invention have the advantage thatoveracylation during the deprotection and radiolabelling is suppressed.

Since the method of the present invention requires fewer steps, andavoids the need for some of the chromatography purification steps of theprior art, it is also both more efficient and more amenable toautomation e.g. using an automated synthesizer apparatus.

A further advantage of the protecting groups of the present invention isthat they are stable at neutral and basic pH and in up to 50% aqueousacetic acid solution, so that the protected precursor can be purifiedusing HPLC chromatography using 0.1% aqueous acetic acid as the mobilephase. Hence, a BTM protected using the approach of the presentinvention can be purified under a variety of conditions which can betailored to the stability of the BTM.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a method forradiolabelling a biological targeting molecule which comprises.

-   -   (i) provision of a protected compound of Formula (IA) or (IB)

[BTM]-X¹   (IA)

Q-[linker]-X¹   (TB)

-   -   (ii) deprotection of the protected compound of Formula (IA) or        (IB) of step (i) to give an aminooxy compound of Formula (IIA)        or (IIB) respectively;

[BTM]-O—NH₂   (IIA)

Q-[linker]-O—NH₂   (IIB)

-   -   (iii) condensation of either:        -   (a) the aminooxy compound of Formula (IIA) with a carbonyl            compound of Formula (IIIA)

Q-[linker]-(C═O)Y¹   (IIIA); or

-   -   (b) the aminooxy compound of Formula (IIB) with a carbonyl        compound of Formula (IIIB),

[BTM]-(C═O)Y¹   (IIIB)

-   -   to give a radiolabelled conjugate of Formula (IVA) or (IVB)        respectively:

[BTM]-O—N=(CY¹)-[linked]-Q   (IVA)

[BTM]-(CY¹)=N—O-[linked]-Q   (IVB)

wherein.

-   -   [BTM] is a biological targeting molecule;    -   X¹ is a protected aminooxy group of formula:

-   -   wherein R¹ and R² are independently chosen from C₁₋₃ alkyl, C₁₋₃        fluoroalkyl or C₄₋₆ aryl;    -   Q is a group which comprises a radioisotope suitable for PET or        SPECT imaging in vivo;    -   Y¹ is H, C₁₋₆ alkyl or C₄₋₁₀ aryl,    -   [linker] is a linker group.

The term “radiolabelling” has its conventional meaning, i.e. a processwherein a radioisotope is covalently attached—in this case to the BTM.

By the term “biological targeting moiety” (BTM) is meant a compoundwhich, after administration, is taken up selectively or localises at aparticular site of the mammalian body in vivo. Such sites may forexample be implicated in a particular disease state or be indicative ofhow an organ or metabolic process is functioning.

The term “deprotection” has its conventional meaning in the field ofchemistry and/or radiochemistry, i.e. the removal of a protecting group.By the term “protecting group” or P^(GP) is meant a group which inhibitsor suppresses undesirable chemical reactions, but which is designed tobe sufficiently reactive that it may be cleaved from the functionalgroup in question under mild enough conditions that do not modify therest of the molecule. After deprotection the desired product isobtained. The use of protecting groups is described in Protective Groupsin Organic Synthesis, 4^(th) Edition, Theorodora W. Greene and Peter G.M. Wuts, [Wiley Blackwell, (2006)].

The term “aminooxy group” has its conventional meaning, and refers to asubstituent of formula —O—NH₂, preferably —CH₂—O—NH₂.

The term “group which comprises a radioisotope” means that either afunctional group comprises the radioisotope, or the radioisotope isattached as an additional species. When a functional group comprises theradioisotope, this means that the chemical structure already containsthe chemical element in question, and the radioactive isotope of thatelement present at a level significantly above the natural abundancelevel of said isotope. Such elevated or enriched levels of isotope aresuitably at least 5 times, preferably at least 10 times, most preferablyat least 20 times; and ideally either at least 50 times the naturalabundance level of the isotope in question, or present at a level wherethe level of enrichment of the isotope in question is 90 to 100%.Examples of such functional groups include fluoroalkyl groups withelevated levels of ¹⁸F, such that the ¹⁸F atom is within the chemicalstructure. When the radioisotope is a radiometal, such as ^(99m)Tc ⁶⁸Gaor ⁶⁴Cu, the “additional species” would typically be a chelating agent.When the radioisotope is radioiodine, then the additional species wouldbe a phenyl or vinyl group, as is known in the art, to stabilise thecarbon-iodine bond to metabolic deiodination in vivo.

The terms “PET” and “SPECT” have their conventional meaning in the fieldof radiopharmaceuticals and refer to Positron Emission Tomography andSingle Photon Emission Tomography respectively.

By the term “linker group” is meant a bivalent group of formula-(A)_(m)- wherein each A is independently —CR₂—, —CR═CR—, —C≡C—,—CR₂CO₂—, —CO₂CR₂—, —NRCO—, —CONR—, —NR(C═O)NR—, —NR(C═S)NR—, —SO₂NR—,—NRSO₂—, —CR₂OCR₂—, —CR₂SCR₂—, —CR₂NRCR₂—, a C₄₋₈ cycloheteroalkylenegroup, a C₄₋₈ cycloalkylene group, a C₅₋₁₂ arylene group, or a C₃₋₁₂heteroarylene group, wherein each R is independently chosen from. H,C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxyalkyl or C₁₋₄hydroxyalkyl;

and m is an integer of value 1 to 20.

In Formula IA, the X¹ group is suitably attached to a functional groupof the BTM as described below.

Preferred Features.

In the first aspect, R¹ and R² are preferably both independently C₁₋₂alkyl. More preferably, R¹ and R² are chosen from methyl and ethyl, mostpreferably R¹ is methyl and R² is ethyl, i.e. an ethoxyethylidine(“Eei”) protecting group.

The method of the first aspect is preferably carried out such that thecompound of Formula (IA) is used in step (i), so that the aminooxycompound of step (ii) is of Formula (IIA), and the radiolabelledconjugate is of Formula (IVA). That is because it is easier toselectively introduce an aminooxy group into a BTM than the carbonyl(i.e. aldehyde or ketone group) of Formula (IIIB).

In the first aspect, Y¹ is preferably H i.e. the carbonyl compound ofFormula (IIIA) or (IIIB) is an aldehyde.

In the first aspect, Q is preferably chosen from ¹⁸F, ¹²³I, ^(99m)Tc,⁶⁸Ga or ⁶⁴Cu. More preferably, Q is ¹⁸F. When Q is ¹⁸F, a preferredcarbonyl compound of Formula (IIIA) is ¹⁸F-4-fluorobenzaldehyde.

After step (iii) of the method of the first aspect, the productconjugate of Formula (IVA) or (IVB) may preferably be separated and/orpurified using standard techniques such as chromatography.

The BTM may be of synthetic or natural origin, but is preferablysynthetic. The term “synthetic” has its conventional meaning, i.e.man-made as opposed to being isolated from natural sources eg. from themammalian body. Such compounds have the advantage that their manufactureand impurity profile can be fully controlled. Monoclonal antibodies andfragments thereof of natural origin are therefore outside the scope ofthe term ‘synthetic’ as used herein. The molecular weight of the BTM ispreferably up to 30,000 Daltons. More preferably, the molecular weightis in the range 200 to 20,000 Daltons, most preferably 300 to 18,000Daltons, with 400 to 16,000 Daltons being especially preferred. When theBTM is a non-peptide, the molecular weight of the BTM is preferably upto 3,000 Daltons, more preferably 200 to 2,500 Daltons, most preferably300 to 2,000 Daltons, with 400 to 1,500 Daltons being especiallypreferred.

BTM preferably comprises: a 3-100 mer peptide, peptide analogue, peptoidor peptide mimetic which may be a linear or cyclic peptide orcombination thereof; a single amino acid; an enzyme substrate, enzymeantagonist enzyme agonist (including partial agonist) or enzymeinhibitor; receptor-binding compound (including a receptor substrate,antagonist, agonist or substrate); oligonucleotides, or oligo-DNA oroligo-RNA fragments. More preferably, BTM comprises either an Affibody™or a single amino acid, a 3-100 mer peptide, an enzyme substrate, anenzyme antagonist an enzyme agonist, an enzyme inhibitor or areceptor-binding compound.

By the term “peptide” is meant a compound comprising two or more aminoacids, as defined below, linked by a peptide bond (ie. an amide bondlinking the amine of one amino acid to the carboxyl of another). Theterm “peptide mimetic” or “mimetic” refers to biologically activecompounds that mimic the biological activity of a peptide or a proteinbut are no longer peptidic in chemical nature, that is, they no longercontain any peptide bonds (that is, amide bonds between amino acids).Here, the term peptide mimetic is used in a broader sense to includemolecules that are no longer completely peptidic in nature, such aspseudo-peptides, semi-peptides and peptoids. The term “peptide analogue”refers to peptides comprising one or more amino acid analogues, asdescribed below. See also Synthesis of Peptides and Peptidomimetics, M.Goodman et al, Houben-Weyl E22c, Thieme.

By the term “amino acid” is meant an L- or D-amino acid, amino acidanalogue (eg. naphthylalanine) or amino acid mimetic which may benaturally occurring or of purely synthetic origin, and may be opticallypure, i.e. a single enantiomer and hence chiral, or a mixture ofenantiomers. Conventional 3-letter or single letter abbreviations foramino acids are used herein. Preferably the amino acids of the presentinvention are optically pure. By the term “amino acid mimetic” is meantsynthetic analogues of naturally occurring amino acids which areisosteres, i.e. have been designed to mimic the steric and electronicstructure of the natural compound. Such isosteres are well known tothose skilled in the art and include but are not limited todepsipeptides, retro-inverso peptides, thioamides, cycloalkanes or1,5-disubstituted tetrazoles [see M. Goodman, Biopolymers, 24, 137,(1985)]. Radiolabelled amino acids such as tyrosine, histidine orproline are known to be useful in vivo imaging agents.

Affibody™ molecules are based on the 58 amino acid residue domainderived from one of the IgG-binding domains of staphylococcal protein A.Affibodies may be used in monomer or dimer form, and have been reviewedby Nygren [FEBS J., 275, 2668-2676 (2008)] and Nilsson et al[Curr.Opin.Drug.Disc.Dev., 10, 167-175 (2007)]. The relatively smallsize of these Affibodies should allow better target tissue penetrationand blood clearance compared to antibodies which are 10 to 20 timeslarger (˜150kDa). Affibodies also have the advantage that they arestable under a range of pH conditions (pH 5.5 to 11).

When the BTM is an enzyme substrate, enzyme antagonist, enzyme agonist,enzyme inhibitor or receptor-binding compound it is preferably anon-peptide, and more preferably is synthetic. By the term “non-peptide”is meant a compound which does not comprise any peptide bonds, ie. anamide bond between two amino acid residues. Suitable enzyme substrates,antagonists, agonists or inhibitors include glucose and glucoseanalogues; fatty acids, or elastase, Angiotensin II or metalloproteinaseinhibitors. Suitable synthetic receptor-binding compounds includeestradiol, estrogen, progestin, progesterone and other steroid hormones;ligands for the dopamine D-1 or D-2 receptor, or dopamine transportersuch as tropanes; and ligands for the serotonin receptor.

The BTM is most preferably a 3-100 mer peptide or peptide analogue. Whenthe BTM is a peptide, it is preferably a 4-30 mer peptide, and mostpreferably a 5 to 28-mer peptide.

When the BTM is an enzyme substrate, enzyme antagonist, enzyme agonistor enzyme inhibitor, preferred such biological targeting molecules ofthe present invention are synthetic, drug-like small molecules i.e.pharmaceutical molecules. Preferred dopamine transporter ligands such astropanes; fatty acids; dopamine D-2 receptor ligands; benzamides;amphetamines; benzylguanidines, iomazenil, benzofuran (IBF) or hippuricacid.

When the BTM is a peptide, preferred such peptides include Peptide A,Peptide B, Peptide C and Peptide D as defined below, as well as:

-   -   somatostatin, octreotide and analogues,    -   peptides which bind to the ST receptor, where ST refers to the        heat-stable toxin produced by E.coli and other micro-organisms;    -   bombesin;    -   vasoactive intestinal peptide;    -   neurotensin;    -   laminin fragments eg. YIGSR, PDSGR, IKVAV, LRE and        KCQAGTFALRGDPQG,    -   N-formyl chemotactic peptides for targeting sites of leucocyte        accumulation,    -   Platelet factor 4 (PF4) and fragments thereof,    -   peptide fragments of α₂-antiplasmin, fibronectin or beta-casein,        fibrinogen or thrombospondin. The amino acid sequences of        α₂-antiplasmin, fibronectin, beta-casein, fibrinogen and        thrombospondin can be found in the following references:        α₂-antiplasmin precursor [M.Tone et al., J.Biochem, 102, 1033,        (1987)]; beta-casein [L.Hansson et al, Gene, 139, 193, (1994)];        fibronectin [A.Gutman et al, FEBS Lett., 207, 145, (1996)];        thrombospondin-1 precursor [V.Dixit et al, Proc. Natl. Acad.        Sci., USA, 83, 5449, (1986)]; R.F.Doolittle, Ann. Rev. Biochem.,        53, 195, (1984);    -   peptides which are substrates or inhibitors of angiotensin, such        as: angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (E. C.        Jorgensen et al, J. Med. Chem., 1979, Vol 22, 9, 1038-1044)    -    [Sar, Ile] Angiotensin II: Sar-Arg-Val-Tyr-Ile-His-Pro-Ile        (R. K. Turker et al., Science, 1972, 177, 1203).    -   Angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu.

More preferred BTM peptides are chosen from Peptide A, Peptide B,Peptide C and Peptide D as defined below:

-   -   (i) Peptide A=an Arg-Gly-Asp peptide;    -   (ii) Peptide B=an Arg-Gly-Asp peptide which comprises the        fragment

-   -   (iii) Peptide C=a c-Met binding cyclic peptide which comprises        the amino acid sequence:

-Cys^(a)-X¹-Cysc-X²-Gly-Pro-Pro-X³-Phe-Glu-Cys^(d)-Trp-Cys^(b)-Tyr-X⁴-X⁵-X⁶-

-   -   wherein X¹ is Asn, His or Tyr;        -   X² is Gly, Ser, Thr or Asn;        -   X³ is Thr or Arg;        -   X⁴ is Ala, Asp, Glu, Gly or Ser;        -   X⁵ is Ser or Thr;        -   X⁶ is Asp or Glu;        -   and Cys^(a-d) are each cysteine residues such that residues            a and b as well as c and d are cyclised to form two separate            disulfide bonds;    -   (iv) Peptide D=a lantibiotic peptide of formula:

Cys^(a)-Xaa-Gln-Ser^(b)-Cys^(c)-Ser^(d)-Phe-Gly-Pro-Phe-Thr^(c)-Phe-Val-Cys^(b)-(HO-Asp)-Gly-Asn-Thr^(a)-Lys^(d)

-   -   wherein Xaa is Arg or Lys;        -   Cys^(a)-Thr^(a), Ser^(b)-Cys^(b) and Cys^(c)-Thr^(c) are            covalently linked via thioether bonds;        -   Ser^(d)-Lys^(d) are covalently linked via a lysinoalanine            bond;        -   HO-Asp is β-hydroxyaspartic acid.

Especially preferred BTM peptides are Peptide B, Peptide C and PeptideD.

A most preferred such Peptide B peptide is of formula (A):

wherein X¹ is either NH₂ or

wherein a is an integer of from 1 to 10.

In Formula A, a is preferably 1.

A preferred c-Met binding cyclic peptide has the sequence:

Ala-Gly-Ser-Cys^(a)-Tyr-Cys^(c)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys^(d)-Trp-Cys^(b)-Tyr-Glu-Thr-Glu-Gly-Thr- Gly-Gly-Gly-Lys.

When the BTM is a peptide, one or both termini of the peptide,preferably both, have conjugated thereto a metabolism inhibiting group(M^(IG)). Having both peptide termini protected in this way is importantfor in vivo imaging applications, since otherwise rapid metabolism wouldbe expected with consequent loss of selective binding affinity for theBTM peptide. By the term “metabolism inhibiting group” (M^(IG)) is meanta biocompatible group which inhibits or suppresses enzyme, especiallypeptidase such as carboxypeptidase, metabolism of the BTM peptide ateither the amino terminus or carboxy terminus. Such groups areparticularly important for in vivo applications, and are well known tothose skilled in the art and are suitably chosen from, for the peptideamine terminus:

N-acylated groups —NH(C═O)R^(G) where the acyl group —(C═O)R^(G) hasR^(G) chosen from: C₁₋₆ alkyl, C₃₋₁₀ aryl groups or comprises apolyethyleneglycol (PEG) building block. Preferred such amino terminusM^(IG) groups are acetyl, benzyloxycarbonyl or trifluoroacetyl, mostpreferably acetyl.

Suitable metabolism inhibiting groups for the peptide carboxyl terminusinclude: carboxamide, tert-butyl ester, benzyl ester, cyclohexyl ester,amino alcohol or a polyethyleneglycol (PEG) building block. A suitableM^(IG) group for the carboxy terminal amino acid residue of the BTMpeptide is where the terminal amine of the amino acid residue isN-alkylated with a C₁₋₄ alkyl group, preferably a methyl group.Preferred such M^(IG) groups are carboxamide or PEG, most preferred suchgroups are carboxamide.

The method of the first aspect is preferably carried out such that steps(ii) and (iii) are carried out simultaneously. In that manner, the freeaminooxy compound of Formula (IIA) or (IIB) is generated in situ, andcan thus react with the carbonyl compound of Formula (IIIA) or (TIB)which is already present in the reaction medium, as it is generated.This is expected to improve the yield of the desired radioconjugateproduct, since the opportunity for side reactions of therelatively-reactive free aminooxy compound is minimized.

The condensation step (iii) of the first aspect is preferably carriedout in the presence of aniline or a salt thereof (e.g. anilinehydrochloride). The use of aniline in oxime ligations has been shown tobe effective in increasing the overall reaction rate and to allow suchreactions to occur at less acidic pH values [Dirksen, et al., Angew.Chem. Int. Ed.Engl., 45, 7581-7584 (2006)].

The method of the first aspect is preferably carried out such that theradiolabelled conjugate of Formula (IVA) or (IVB) is obtained in a formsuitable for mammalian administration, more preferably in a formsuitable for use as a radiopharmaceutical for in vivo imaging asdescribed in the second aspect (below).

The method of the first aspect is preferably carried out using anautomated synthesizer apparatus as described in the second and fourthaspects (below).

N-(1-Ethoxyethylidene)-2-aminooxyacetic acid N-hydroxysuccinimidyl esteris commercially available from Iris Biotech GmbH (Waldershofer Str.49-51, 95615 Marktredwitz, Germany). That Eei-protected amino-oxy activeester can be conjugated directly to an amine-containing BTM (eg. havinga Lys residue), to give a protected compound of Formula (IA). Furtherroutes to Eei-protected peptides are described by Dulery et al[Tetrahedron, 63, 11952-11958 (2007)] and Foillard et al [J.Org.Chem.,73, 983-991 (2008)]. Dulery and Foillard also describe suitableconditions for deprotection of the Eei protecting group.

The aminooxy compound of Formula IB can be obtained as follows.(4-aminophenyl)trimethylammonium andN-(1-ethoxyethylidene)-2-aminooxyacetic acid N-hydroxysuccinimidyl esterare reacted in an organic solvent in the presence of tertiary base toform(Z)-4-(2-4(1-ethoxyethylidene)amino)oxy)acetamido)-N,N,N-trimethylbenzenaminium.(Z)-4-(2-(((1-ethoxyethylidene)amino)oxy)acetamido)-N,N,N-trimethylbenzenaminiumis labelled with ¹⁸F using standard ¹⁸F-labelling conditions to afford(Z)-ethyl N-2-((4-fluorophenyl)amino)-2-oxoethoxyacetimidate:

The carbonyl compound of Formula IIIA can be obtained as follows. The¹⁸F-fluorinated aldehyde may be ¹⁸F-fluorobenzaldehyde orp-(di-tert-butyl-¹⁸F-fluorosilypbenzaldehyde (¹⁸F-SiFA-A). ¹⁸F-labelledaliphatic aldehydes of formula ¹⁸F(CH₂)₂O[CH₂CH₂O]_(q)CH₂CHO, where q is3, can be obtained by the method of Glaser et al [Bioconj.Chem., 19(4),951-957 (2008)]. ¹⁸F-fluorobenzaldehyde can be obtained by the method ofGlaser et al [J.Lab.Comp.Radiopharm., 52, 327-330 (2009)]. The precursorto ¹⁸F-fluorobenzaldehyde, i.e. Me₃N⁺—C₆H₄—CHO. CF₃SO₃ ⁻can be obtainedby the method of Haka et al [J.Lab.Comp.Radiopharm., 27, 823-833(1989)].

¹²³I-benzaldehyde can be prepared from the corresponding trimethyltinprecursor, which is described by Thumshirn et al [Chem.Eur.J., 9,2717-2725 (2003)].

When the BTM is a monoclonal antibody, the carbonyl group of FormulaIIIB can be introduced by oxidation of the sugar moiety of the antibodyusing e.g. periodate [Kurth et al and references therein J.Med.Chem.,36, 1255-1261 (1993)]. Amino acids having aldehyde side-chains can beintroduced into peptide sequences by the method described in Tet.Lett.,43(12), p. 2303-2306 (2002).

The carbonyl compound of Formula IIIA or IIIB may optionally begenerated in situ by deprotection of a suitable protected derivative.The use of carbonyl protecting groups is described in Protective Groupsin Organic Synthesis, 4^(th) Edition, Theorodora W. Greene and Peter G.M. Wuts, [Wiley Blackwell, (2006)].

In a second aspect, the present invention provides a method ofpreparation of a radiopharmaceutical composition, wherein saidradiopharmaceutical composition comprises the radiolabelled conjugate ofFormula (IVA) or (IVB) as defined in the first aspect, together with abiocompatible carrier in a form suitable for mammalian administration,and said method of preparation comprises the radiolabelling method ofthe first aspect.

Preferred embodiments of the radiolabelled conjugate of Formula (IVA) or(IVB) in the second aspect are as described in the first aspect (above).The radiolabelled conjugate of the second aspect is preferably ofFormula (IVA).

By the phrase “in a form suitable for mammalian administration” is meanta composition which is sterile, pyrogen-free, lacks compounds whichproduce toxic or adverse effects, and is formulated at a biocompatiblepH (approximately pH 4.0 to 10.5). Such compositions lack particulateswhich could risk causing emboli in vivo, and are formulated so thatprecipitation does not occur on contact with biological fluids (e.g.blood). Such compositions also contain only biologically compatibleexcipients, and are preferably isotonic.

The “biocompatible carrier” is a fluid, especially a liquid, in whichthe radioconjugate can be suspended or preferably dissolved, such thatthe composition is physiologically tolerable, i.e. can be administeredto the mammalian body without toxicity or undue discomfort. Thebiocompatible carrier is suitably an injectable carrier liquid such assterile, pyrogen-free water for injection; an aqueous solution such assaline (which may advantageously be balanced so that the final productfor injection is isotonic); an aqueous buffer solution comprising abiocompatible buffering agent (e.g. phosphate buffer); an aqueoussolution of one or more tonicity-adjusting substances (e.g. salts ofplasma cations with biocompatible counterions), sugars (e.g. glucose orsucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g.glycerol), or other non-ionic polyol materials (e.g.polyethyleneglycols, propylene glycols and the like). Preferably thebiocompatible carrier is pyrogen-free water for injection, isotonicsaline or phosphate buffer.

The radiolabelled conjugate and biocompatible carrier are supplied in asuitable vial or vessel which comprises a sealed container which permitsmaintenance of sterile integrity and/or radioactive safety, plusoptionally an inert headspace gas (eg. nitrogen or argon), whilstpermitting addition and withdrawal of solutions by syringe or cannula. Apreferred such container is a septum-sealed vial, wherein the gas-tightclosure is crimped on with an overseal (typically of aluminium). Theclosure is suitable for single or multiple puncturing with a hypodermicneedle (e.g. a crimped-on septum seal closure) whilst maintainingsterile integrity. Such containers have the additional advantage thatthe closure can withstand vacuum if desired (eg. to change the headspacegas or degas solutions), and withstand pressure changes such asreductions in pressure without permitting ingress of externalatmospheric gases, such as oxygen or water vapour.

Preferred multiple dose containers comprise a single bulk vial (e.g. of10 to 50 cm³ volume) which contains multiple patient doses, wherebysingle patient doses can thus be withdrawn into clinical grade syringesat various time intervals during the viable lifetime of the preparationto suit the clinical situation. Pre-filled syringes are designed tocontain a single human dose, or “unit dose” and are therefore preferablya disposable or other syringe suitable for clinical use.

The radiopharmaceutical composition may contain additional optionalexcipients such as: an antimicrobial preservative, pH-adjusting agent,filler, radioprotectant, solubiliser or osmolality adjusting agent. Bythe term “radioprotectant” is meant a compound which inhibitsdegradation reactions, such as redox processes, by trappinghighly-reactive free radicals, such as oxygen-containing free radicalsarising from the radiolysis of water. The radioprotectants of thepresent invention are suitably chosen from: ascorbic acid,para-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e.2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cationas described above. By the term “solubiliser” is meant an additivepresent in the composition which increases the solubility of the imagingagent in the solvent. A preferred such solvent is aqueous media, andhence the solubiliser preferably improves solubility in water. Suitablesuch solubilisers include: C₁₋₄ alcohols; glycerine; polyethylene glycol(PEG); propylene glycol; polyoxyethylene sorbitan monooleate; sorbitanmonooloeate; polysorbates;poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics™); cyclodextrins (e.g. alpha, beta or gamma cyclodextrin,hydroxypropyl-β-cyclodextrin or hydroxypropyl-γ-cyclodextrin) andlecithin.

By the term “antimicrobial preservative” is meant an agent whichinhibits the growth of potentially harmful micro-organisms such asbacteria, yeasts or moulds. The antimicrobial preservative may alsoexhibit some bactericidal properties, depending on the dosage employed.The main role of the antimicrobial preservative(s) of the presentinvention is to inhibit the growth of any such micro-organism in thepharmaceutical composition. The antimicrobial preservative may, however,also optionally be used to inhibit the growth of potentially harmfulmicro-organisms in one or more components of kits used to prepare saidcomposition prior to administration. Suitable antimicrobialpreservative(s) include: the parabens, i.e. methyl, ethyl, propyl orbutyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol;cetrimide and thiomersal. Preferred antimicrobial preservative(s) arethe parabens.

The term “pH-adjusting agent” means a compound or mixture of compoundsuseful to ensure that the pH of the composition is within acceptablelimits (approximately pH 4.0 to 10.5) for human or mammalianadministration. Suitable such pH-adjusting agents includepharmaceutically acceptable buffers, such as tricine, phosphate or TRIS[i.e. tris(hydroxymethyl)aminomethane], and pharmaceutically acceptablebases such as sodium carbonate, sodium bicarbonate or mixtures thereof.When the composition is employed in kit form, the pH adjusting agent mayoptionally be provided in a separate vial or container, so that the userof the kit can adjust the pH as part of a multi-step procedure.

By the term “filler” is meant a pharmaceutically acceptable bulkingagent which may facilitate material handling during production andlyophilisation. Suitable fillers include inorganic salts such as sodiumchloride, and water soluble sugars or sugar alcohols such as sucrose,maltose, mannitol or trehalose.

The method of second aspect may be carried out in various ways:

-   -   a) aseptic manufacture techniques in which the steps are carried        out in a clean room environment;    -   b) terminal sterilisation, in which steps (i)-(iii) of the first        aspect are carried out without using aseptic manufacture, and        then sterilised as the last step [eg. by gamma irradiation,        autoclaving dry heat or chemical treatment (e.g. with ethylene        oxide)];    -   c) kit methodology in which a sterile, non-radioactive kit        formulation comprising a suitable non-radioactive precursor of        Formula (IA) or (TIB) and optional excipients is reacted with        the radioactive compound of Formula (IIIA) or (IB) respectively;    -   d) aseptic manufacture techniques in which the steps are carried        out using an automated synthesizer apparatus.

Method (d) is preferred. That is described in the fourth aspect (below).

In a third aspect, the present invention provides a precursor useful inthe method of the second aspect, wherein said precursor comprises theprotected compound of Formula (IA) in sterile form.

Preferred embodiments of the compound of Formula (IA) in the thirdaspect are as described in the first aspect (above).

The “precursor” of Formula (IA) comprises a non-radioactive derivativeof the BTM, Such precursors are synthetic and can conveniently beobtained in good chemical purity. The “precursor” may optionally furthercomprise one or more protecting groups (P^(GP)) for certain functionalgroup(s) of the biological targeting molecule. The P^(GP) is as definedin the first aspect (above).

A preferred sterile form of the protected compound of Formula (IA) is alyophilised solid. More preferably, the sterile precursor is supplied aspart of a cassette as is described in the fifth aspect (below).

Methods of obtaining sterile compounds are as described for theradiopharmaceutical composition of the second aspect (above).

In a fourth aspect, the present invention provides the use of anautomated synthesizer apparatus to carry out the method of the first orsecond aspects.

Preferred embodiments of the method of the first aspect in the thirdaspect are as described in the first aspect (above). Preferably, thefourth aspect is used to carry out the method of the second aspect, i.e.to prepare a radiopharmaceutical composition.

By the term “automated synthesizer” is meant an automated module basedon the principle of unit operations as described by Satyamurthy et al[Clin.Positr.Imag., 2(5), 233-253 (1999)]. The term ‘unit operations’means that complex processes are reduced to a series of simpleoperations or reactions, which can be applied to a range of materials.Such automated synthesizers are preferred for the method of the presentinvention especially when a radiopharmaceutical composition is desired.They are commercially available from a range of suppliers [Satyamurthyet al, above], including: GE Healthcare; CTI Inc; Ion Beam ApplicationsS.A. (Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest(Germany) and Bioscan (USA).

Commercial automated synthesizers also provide suitable containers forthe liquid radioactive waste generated as a result of theradiopharmaceutical preparation Automated synthesizers are not typicallyprovided with radiation shielding, since they are designed to beemployed in a suitably configured radioactive work cell. The radioactivework cell provides suitable radiation shielding to protect the operatorfrom potential radiation dose, as well as ventilation to remove chemicaland/or radioactive vapours. The automated synthesizer preferablycomprises a cassette.

By the term “cassette” is meant a piece of apparatus designed to fitremovably and interchangeably onto an automated synthesizer apparatus(as defined above), in such a way that mechanical movement of movingparts of the synthesizer controls the operation of the cassette fromoutside the cassette, i.e. externally. Suitable cassettes comprise alinear array of valves, each linked to a port where reagents or vialscan be attached, by either needle puncture of an inverted septum-sealedvial, or by gas-tight, marrying joints. Each valve has a male-femalejoint which interfaces with a corresponding moving arm of the automatedsynthesizer. External rotation of the arm thus controls the opening orclosing of the valve when the cassette is attached to the automatedsynthesizer. Additional moving parts of the automated synthesizer aredesigned to clip onto syringe plunger tips, and thus raise or depresssyringe barrels.

The cassette is versatile, typically having several positions wherereagents can be attached, and several suitable for attachment of syringevials of reagents or chromatography cartridges (eg. solid phaseextraction or SPE). The cassette always comprises a reaction vessel.Such reaction vessels are preferably 1 to 10 cm³, most preferably 2 to 5cm³ in volume and are configured such that 3 or more ports of thecassette are connected thereto, to permit transfer of reagents orsolvents from various ports on the cassette. Preferably the cassette has15 to 40 valves in a linear array, most preferably 20 to 30, with 25being especially preferred. The valves of the cassette are preferablyeach identical, and most preferably are 3-way valves. The cassettes aredesigned to be suitable for radiopharmaceutical manufacture and aretherefore manufactured from materials which are of pharmaceutical gradeand ideally also are resistant to radiolysis.

Preferred automated synthesizers of the present invention comprise adisposable or single use cassette which comprises all the reagents,reaction vessels and apparatus necessary to carry out the preparation ofa given batch of radi ° fluorinated radiopharmaceutical The cassettemeans that the automated synthesizer has the flexibility to be capableof making a variety of different radiopharmaceuticals with minimal riskof cross-contamination, by simply changing the cassette. The cassetteapproach also has the advantages of: simplified set-up hence reducedrisk of operator error; improved GMP (Good Manufacturing Practice)compliance; multi-tracer capability; rapid change between productionruns; pre-run automated diagnostic checking of the cassette andreagents; automated barcode cross-check of chemical reagents vs thesynthesis to be carried out; reagent traceability; single-use and henceno risk of cross-contamination, tamper and abuse resistance.

In a fifth aspect, the present invention provides a single-use,disposable cassette suitable for use in the automated synthesizer asdefined in the fourth aspect, wherein said cassette comprises theprecursor of the third aspect.

Preferred aspects of the automated synthesizer and cassette in the fifthaspect are as described in the fourth aspect (above) Preferred aspectsof the precursor in the fifth aspect are as described in the thirdaspect (above).

In a sixth aspect, the present invention provides a protected compoundof Formula (IA), wherein X¹ and preferred aspects thereof are as definedin the first aspect, and the BTM is an Affibody, or Peptide A, PeptideB, Peptide C or Peptide D, and preferred aspects thereof, as defined inthe first aspect (above).

In a seventh aspect, the present invention provides a protected compoundof Formula (1B), wherein X^(l) and Q and preferred aspects thereof areas defined in the first aspect.

In an eighth aspect, the present invention provides the use of theprotected compound of Formula (IA) or (IB) as defined in the firstaspect, in the radiolabelling of a biological targeting molecule (BTM)as defined in the first aspect. Preferred aspects of the compound ofFormula (IA) or (IB) and BTM in the eighth aspect, are as described inthe first aspect (above).

The invention is illustrated by the non-limiting Examples detailedbelow. Example 1 provides the synthesis of a c-Met targeting peptide ofthe invention (“Peptide 1”). Example 1 provides the synthesis of anaminooxy-functionalised Peptide 1 (“Compound 1”), wherein the aminooxyfunctional group is protected with a protecting group of the invention(Eei). Example 3 provides the deprotection of Compound 1 to give thefree aminooxy-Peptide 1 (“Compound 2”). Example 4 provides the in situ(i.e. one-pot) deprotection and aldehyde conjugation of Compound 1 togive the conjugate (“Compound 3”). Example 5 provides the synthesis of afurther Eei-protected peptide of the invention, and Example 6 itsdeprotection.

Abbreviations.

Conventional single letter or 3-letter amino acid abbreviations areused.

Ac: Acetyl

Acm: Acetamidomethyl

ACN: Acetonitrile

AcOH: Acetic acid.

Boc: tert-Butyloxycarbonyl

tBu: tertiary-butyl

DCM: Dichloromethane

DIPEA: N,N-Diisopropylethyl amine

DMF: Dimethylformamide

DMSO: Dimethylsulfoxide

Eei: ethoxyethylidine;

Fmoc: 9-Fluorenylm ethoxy carbonyl

HBTU: 0-Benzotriazol-1-yl-N,N,N′ -tetram ethy luroniumhexafluorophosphate

HPLC: High performance liquid chromatography

NHS: N-hydroxy-succinimide

NMM: N-Methylmorpholine

NMP: 1-Methyl-2-pyrrolidinone

Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl

tBu: tert-butyl

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

TIS: Triisopropylsilane

Trt: Trityl.

COMPOUNDS OF THE INVENTION

Name Structure Peptide 1 Disulfide bridges at Cys4-16 and Cys6-14; Ac-Ala-Gly-Ser-Cys-Tyr- Cys-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly- Thr-Gly-Gly-Gly-Lys-NH₂ orAc-AGSCYCSGPPRFECWCYETEGTGGGK-NH₂ Compound 1 [Peptide 1]-NH(CO)-(CH₂)-O-N = C(CH₃)(OCH₂CH₃) Compound 2 [Peptide 1]-NH(CO)-(CH₂)- O-NH₂Compound 3 [Peptide 1]-NH(CO)-(CH₂)- O-NH = CH(CH₃) Compound 4[LBP1]-(CO)CH₂ONC(CH₃)OEt. LBP1 = Cys^(a)-Lys-Gln-Ser^(b)-Cys^(c)-Ser^(d)-Phe-Gly-Pro-Phe-Thr^(c)-Phe- Val-Cys^(b)-(HO-Asp)-Gly-Asn-Thr^(a)-Lys^(d) (Mixture of isomers LBP1functionalized at either Cys^(a) or Xaa Lys groups). Compound 5[LBP1]-(CO)CH₂ONH₂ (Mixture of isomers LBP1functionalized at either Cys^(a) or Xaa Lys groups).

where:

Compounds 1, 2 and 3 are functionalised at the epsilon amine group ofthe carboxy terminal Lys of Peptide 1.

Example 1 Synthesis of Peptide 1.

Step (a). Synthesis of Protected Precursor Linear Peptide.

The precursor linear peptide has the structure:

Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂

The peptidyl resinH-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-Glu(OtBu)-Thr(ψ^(Me,Me)pro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-Polymerwas assembled on an Applied Biosystems 433A peptide synthesizer usingFmoc chemistry starting with 0.1 mmol Rink Amide Novagel resin. Anexcess of 1 mmol pre-activated amino acids (using HBTU) was applied inthe coupling steps. Glu-Thr pseudoproline (Novabiochem 05-20-1122) wasincorporated in the sequence. The resin was transferred to a nitrogenbubbler apparatus and treated with a solution of acetic anhydride (1mmol) and NMM (1 mmol) dissolved in DCM (5 mL) for 60 min. The anhydridesolution was removed by filtration and the resin washed with DCM anddried under a stream of nitrogen.

The simultaneous removal of the side-chain protecting groups andcleavage of the peptide from the resin was carried out in TFA (10 mL)containing 2.5% TIS, 2.5% 4-thiocresol and 2.5% water for 2 hours and 30min. The resin was removed by filtration, TFA removed in vacuo anddiethyl ether added to the residue. The formed precipitate was washedwith diethyl ether and air-dried affording 264 mg of crude peptide.

Purification by preparative HPLC (gradient: 20-30% B over 40 min whereA=H₂O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column:Phenomenex Luna 5μ C18 (2) 250×21.20 mm, detection: UV 214 nm, productretention time: 30 min) of the crude peptide afforded 100 mg of purePeptide 1 linear precursor. The pure product was analysed by analyticalHPLC (gradient: 10-40% B over 10 min where A=H₂O/0.1% TFA and B=ACN/0.1%TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3μ C18 (2) 50×2 mm,detection: UV 214 nm, product retention time: 6.54 min). Further productcharacterisation was carried out using electrospray mass spectrometry(MH₂ ²⁺ calculated: 1464.6, MH₂ ²⁺ found: 1465.1).

Step (b): Formation of Monocvclic Cvs4-16 Disulfide Bridge.

Cys4-16; Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂

The linear precursor from step (a) (100 mg) was dissolved in 5%DMSO/water (200 mL) and the solution adjusted to pH 6 using ammonia. Thereaction mixture was stirred for 5 days. The solution was then adjustedto pH 2 using TFA and most of the solvent removed by evaporation invacuo. The residue (40 mL) was injected in portions onto a preparativeHPLC column for product purification.

Purification by preparative HPLC (gradient: 0% B for 10 min, then 0-40%B over 40 min where A=H₂O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10mL/min, column: Phenomenex Luna 5μ C18 (2) 250×21.20 mm, detection: UV214 nm, product retention time: 44 min) of the residue afforded 72 mg ofpure Peptide 1 monocyclic precursor. The pure product (as a mixture ofisomers P1 to P3) was analysed by analytical HPLC (gradient: 10-40% Bover 10 min where A=H₂O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3mL/min, column: Phenomenex Luna 3μ C18 (2) 50×2 mm, detection: UV 214nm, product retention time: 5.37 min (P1); 5.61 min (P2); 6.05 min(P3)). Further product characterisation was carried out usingelectrospray mass spectrometry (MH₂ ²⁺ calculated: 1463.6, MH₂ ²⁺ found:1464.1 (P1); 1464.4 (P2); 1464.3 (P3)).

Step (c): Formation of Second Cys6-14 Disulfide Bridge (Peptide 1).

The monocyclic precursor from step (b) (72 mg) was dissolved in 75%AcOH/water (72 mL) under a blanket of nitrogen. 1 M HCl (7.2 mL) and0.05 M I₂ in AcOH (4.8 mL) were added in that order and the mixturestirred for 45 min. 1 M ascorbic acid (1 mL) was added giving acolourless mixture. Most of the solvents were evaporated in vacuo andthe residue (18 mL) diluted with water/0.1 TFA (4 mL) and the productpurified using preparative HPLC. Purification by preparative HPLC(gradient: 0% B for 10 min, then 20-30% B over 40 min where A=H₂O/0.1%TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5μC18 (2) 250×21.20 mm, detection: UV 214 nm, product retention time:43-53 min) of the residue afforded 52 mg of pure Peptide 1. The pureproduct was analysed by analytical ACN/0.1% TFA, flow rate: 0.3 mL/min,column: Phenomenex Luna 3μ C18 (2) 50×2 mm, detection: UV 214 nm,product retention time: 6.54 min). Further product characterisation wascarried out using electrospray mass spectrometry (MH₂ ²⁺ calculated:1391.5, MH₂ ²⁺ found: 1392.5).

Example 2 Synthesis of Eei-protected Aminooxy Conjugate of Peptide 1(Compound 1).

Peptide 1 (0.60 g, 0.22 mmol) and Eei-AOAc-Osu (IRIS Biotech; 83 mg,0.32 mmol) were dissolved in DMF (5 mL). DIPEA (75 μL, 2 mmol) was addedand the reaction mixture shaken for 30 min. A second aliquot of DIPEA(75 μL, 2 mmol) was added and the reaction mixture shaken for 1 hr. Thereaction mixture was then diluted with 10% ACN/water/0.1% ammoniumacetate (10 mL), and the product purified using preparative HPLC. Thefractions containing pure product were combined and the productlyophilised affording 550 mg (89% yield) of Compound 1.

The pure product was analysed by analytical HPLC (gradient: 10-40% Bover 5 min where A=H₂O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.6mL/min, column: Phenomenex Luna 3μ C18 (2) 20×2 mm, detection: UV 214nm, product retention time: 3.56 min). Further product characterisationwas carried out using electrospray mass spectrometry (MH22+ calculated:1463.1, MH22+ found: 1463.2).

Example 3 Deprotection to Give the Aminooxv Conjugate of Peptide 1(Compound 2).

Compound 1 (Example 2; 461 mg. 158 μmol) was suspended in a solution of2.5% TFA/water (46 mL) and the solution shaken/sonicated for 60 min. Thesuspension was diluted with water (414 mL) and the solution freeze-driedaffording 472 mg (105%) Compound 2.

The pure product was analysed by analytical HPLC (gradient: 10-40% Bover 5 min where A=H₂O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.6mL/min, column: Phenomenex Luna 3μ C18 (2) 20×2 mm, detection: UV 214nm, product retention time: 3.00 min). Further product characterisationwas carried out using electrospray mass spectrometry (MH22+ calculated:1428.1, MH22+ found: 1428.6).

Example 4 One Pot Conjugation of Compound 1 with an Aldehyde.

Acetaldehyde (1 μL, 17 μmol) in ethanol (0.5 mL) was added to a mixtureof Compound 1 (Example 2; 5 mg, 1.7 μmol) in 1 M HCl (0.5 mL) and thereaction mixture shaken/sonicated for 30 min. The reaction mixture wasthen diluted with 10% ACN/water/0.1% TFA (7 mL) and the product purifiedby semi-preparative HPLC affording 3.8 mg (78%) of Compound 3.

The pure product was analysed by analytical HPLC (gradient: 10-40% Bover 5 min where A=H₂O/0.1 TFA and B=ACN/0.1% TFA, flow rate: 0.6mL/min, column: Phenomenex Luna 3μ C18 (2) 20×2 mm, detection: UV 214nm, product retention time: 3.22 min). Further product characterisationwas carried out using electrospray mass spectrometry (MH22+ calculated:1441.1, MH22+ found: 1441.4).

Example 5 Synthesis of (Eei-aminooxy)aeetyl-Duramycin (Compound 4).

Duramycin (Sigma-Aldrich, 50 mg, 25 imol), (Eei-aminooxy)acetic acid NHSester (Iris Biotech., 5.1 mg, 20 μmol) and DIPEA (17 μL, 100 μmol) weredissolved in NMP (1 mL). The reaction mixture was shaken for 45 min. Themixture was then diluted with water/0.1% acetic acid (8 mL) and theproduct purified using preparative HPLC

Purification by preparative HPLC (as for Example 1 with gradient 14-45%B over 40 min where A=water/0.1% acetic acid and B=ACN) afforded 14 mgpure Compound 4 (yield 26%). The purified material was analysed by LC-MS(gradient: 20-50% B over 5 min, t_(R): 2.5 and 2.7 min, found m/z:1078.8, expected MH₂ ²⁺: 1078.5).

Chromatographic resolution of the (Eei-aminooxy)acetyl-Duramycinregioisomers could be achieved on analytical HPLC using 0.1% TFA.However, the Eei protecting group is labile in 0.1% TFA so preparativeseparation was not feasible. The regioisomers were not resolved using0.1% acetic acid.

Example 6: Synthesis of Aminooxyacetyl-Duramycin (Compound 5).

Compound 4 (Example 5; 14 mg) was treated with 2.5% TFA/water (2.8 mL)under argon for 40 min. The reaction mixture was diluted with water (31mL) and the product lyophilized (frozen under argon usingisopropanol/dry-ice) affording 18 mg Compound 5. The lyophilized productwas analysed by LC-MS (gradient: 20-50% B over 5 min, t_(R): 2.5 and 2.1min, found m/z: 1043.8, expected MH₂ ²⁺: 1043.5).

Chromatographic resolution of the Compound 5 regioisomers could beachieved on analytical HPLC using 0.1% TFA. However, due to the highreactivity of the free aminooxy group towards traces of ketones andaldehydes in the solvent and the atmosphere, no attempt was made toseparate the regioisomers.

What is claimed:
 1. A method for radiolabelling a biological targetingmolecule which comprises: (i) providing a compound of Formula (IB)Q-[linker]-X¹   (IB) (ii) deprotection of the protected compound ofFormula (IB) of step (i) to give an aminooxy compound of Formula (IIB)respectivelyQ-[linker]-O—NH₂   (IIB) (iii) condensation of the aminooxy compound ofFormula (IIB) with a carbonyl compound of Formula (IIIB),[BTM]-(C═O)Y¹   (IIIB) to give a radiolabelled conjugate of Formula(IVB):[BTM]-(CY¹)═N—O-[linker]Q   (IVB) wherein: [BTM] is a biologicaltargeting molecule; X¹ is a protected aminooxy group of formula:

wherein R¹ and R² are independently chosen from C₁₋₃ alkyl, C₁₋₃fluoroalkyl or C₄₋₆ aryl; Q is a group which comprises a radioisotopesuitable for PET or SPECT imaging in vivo; Y¹ is H, C₁₋₆ alkyl or C₄₋₁₀aryl, [linker] is a linker group.
 2. The method of claim 1, where R¹ andR² are independently C₁₋₃ alkyl.
 3. The method of claim 1, where Y¹ isH.
 4. The method of claim 1, where Q is chosen from ¹⁸F, ¹²³I, ^(99m)TC,⁶⁸Ga or ⁶⁴Cu.
 5. The method of claim 4, where Q is ¹⁸F.
 6. The method ofclaim 1, where the BTM comprises a single amino acid, a 3-100 merpeptide, an enzyme substrate, an enzyme antagonist an enzyme agonist, anenzyme inhibitor or a receptor-binding compound.
 7. The method of claim6, where the BTM comprises an Affibody™.
 8. The method of claim 6, wherethe BTM comprises a 3-100 mer peptide which is chosen from Peptide A,Peptide B, Peptide C and Peptide D.
 9. The method of claim 8, whereinPeptide A is an Arg-Gly-Asp peptide.
 10. The method of claim 8, whereinPeptide B is an Arg-Gly-Asp peptide which comprises the fragment


11. The method of claim 8, wherein Peptide B is of formula (A):

wherein X¹ is either —NH2 or

wherein a is an integer of from 1 to
 10. 12. The method of claim 8,wherein Peptide B is a Fluciclatide molecule:


13. The method of claim 8, wherein Peptide C is a c-Met binding cyclicpeptide which comprises the amino acid sequence:-Cys^(a)-X¹-Cys^(c)-X²-Gly-Pro-Pro-X³-Phe-Glu-Cys^(d)-Trp-Cys^(b)-Tyr-X⁴-X⁵-X⁶-

Wherein X¹ is Asn, His or Tyr; X² is Gly, Ser, Thr or Asn; X³ is Thr orArg; X⁴ is Ala, Asp, Glu, Gly or Ser; X⁵ is Ser or Thr; X⁶ is Asp orGlu; and Cys^(a-d) are each cysteine residues such that residues a and bas well as c and d are cyclised to form two separate disulfide bonds.14. The method of claim 8, wherein Peptide D is a lantibiotic peptide offormula:Cys^(a)-Xaa-Gln-Ser^(b)-Cys^(c)-Ser^(d)-Phe-Gly-Pro-Phe-Thr^(c)-Phe-Val-Cys^(b)-(HO-Asp)-Gly-Asn-Thr^(a)-Lys^(d)

wherein Xaa is Arg or Lys; Cys^(a)-Thr, Ser^(b)-Cys^(b) andCys^(c)-Thr^(c) are covalently linked via thioether bonds;Ser^(d)-Lys^(d) are covalently linked via a lysinoalanine bond; HO-Aspis β-hydroxyaspartic acid.
 15. The method of claim 1, where steps (ii)and (iii) are carried out simultaneously.
 16. The method of claim 1,where the condensation step (iii) is carried out in the presence ofaniline.
 17. The method of claim 1, which is carried out using anautomated synthesizer apparatus.
 18. The method of claim 17, whereinsaid automated synthesizer comprises a single-use, disposable cassette.19. A method of preparation of a radiopharmaceutical composition,wherein said radiopharmaceutical composition comprises the radiolabelledconjugate of Formula (IVB) as defined in claim 1, together with abiocompatible carrier in a form suitable for mammalian administration,and said method of preparation comprises the radiolabelling method ofclaim
 1. 20. A protected compound of Formula (IB)Q-[linker]-X¹   (IB) wherein Xis a protected aminooxy group of formula:

wherein R¹ and R² are independently chosen from C₁₋₃ alkyl, C₁₋₃fluoroalkyl or C₄₋₆ aryl; and Q is a group which comprises aradioisotope suitable for PET or SPECT imaging in vivo.